Peptidomimetic macrocycles
Biologically active crosslinked polypeptides with improved properties relative to their corresponding precursor polypeptides, having good cell penetration properties and reduced binding to human proteins, and methods of identifying and making such improved polypeptides are described.
1. A method of screening for an enhanced alpha-helical polypeptide with enhanced cellular efficacy in human whole blood, the enhanced alpha-helical polypeptide comprising a cross-linker connecting a first amino acid and a second amino acid of the polypeptide, the method comprising:
(a) providing a sequence of a parent polypeptide comprising a cross-linker connecting a first amino acid and a second amino acid of the parent polypeptide sequence,
(b) producing a modified alpha-helical polypeptide having the same sequence as the parent polypeptide sequence except that at least one amino acid side chain that is not essential for target binding is different in comparison to the alpha-helical parent polypeptide sequence, wherein the modified alpha-helical polypeptide comprises a cross-linker connecting a first amino acid and a second amino acid of the modified alpha-helical polypeptide;
(c) measuring an in vitro activity (EC 50 ) of the modified alpha-helical polypeptide in a whole cell assay wherein activity is mediated by binding to a target, in the presence and absence of human serum;
(d) determining an apparent affinity (K d *) of the modified alpha-helical polypeptide for human serum proteins; and
(e) selecting the modified alpha-helical polypeptide as an enhanced alpha-helical polypeptide if the modified alpha-helical polypeptide has a K d * between about 1 and 700 micromolar.
2. The method of claim 1 , wherein the enhanced alpha-helical polypeptide has a K d * between about 1 and 70 micromolar.
3. The method of claim 1 , wherein the enhanced alpha-helical polypeptide has a K d * of about 1-10 micromolar.
4. The method of claim 1 , wherein the enhanced alpha-helical polypeptide possesses an estimated free fraction in human blood of about 0.1-50%, wherein the estimated free fraction is defined by the equation
FreeFraction
=
K
d
*
K
d
*
+
[
HSA
]
total
and [HSA] total is 700 micromolar.
5. The method of claim 1 , wherein the enhanced alpha-helical polypeptide possesses an estimated free fraction in human blood of about 0.5-10%.
6. The method of claim 1 , wherein at least one of the first and second amino acids is an α,α-disubstituted amino acid.
7. The method of claim 1 , wherein both the first and second amino acids are α,α-disubstituted.
8. The method of claim 1 , wherein the enhanced alpha-helical polypeptide comprises 1 or 2 turns of an alpha-helix.
9. The method of claim 1 , wherein the first amino acid and the second amino acid are separated by three amino acids.
10. The method of claim 1 , wherein the cross-linker comprises between 6 and 14 consecutive bonds.
11. The method of claim 1 , wherein the cross-linker comprises between 8 and 12 consecutive bonds.
12. The method of claim 1 , wherein the enhanced alpha-helical polypeptide comprises a ring of about 18 atoms to 26 atoms.
13. The method of claim 1 , wherein the first amino acid and the second amino acid are separated by six amino acids.
14. The method of claim 1 , wherein the cross-linker comprises between 8 and 16 consecutive bonds.
15. The method of claim 1 , wherein the cross-linker comprises between 10 and 13 consecutive bonds.
16. The method of claim 1 , wherein the enhanced alpha-helical polypeptide comprises a ring of about 29 atoms to 37 atoms.
17. The method of claim 1 , wherein the length of the cross-linker is about 5 Å to about 9 Å per turn of an alpha-helix.
18. The method of claim 1 , wherein the enhanced alpha-helical polypeptide carries a net positive charge at pH 7.4.
19. The method of claim 1 , wherein the enhanced alpha-helical polypeptide comprises one or more of a halogen, an alkyl group, a fluorescent moiety, an affinity label, a targeting moiety, or a radioisotope.
20. The method of claim 1 , wherein the enhanced alpha-helical polypeptide provides a therapeutic effect.
21. The method of claim 1 , wherein the enhanced alpha-helical polypeptide possesses an apparent affinity to human serum proteins of about 1 micromolar or weaker.
22. The method of claim 1 , wherein the enhanced alpha-helical polypeptide possesses an apparent affinity to human serum proteins of about 3 micromolar or weaker.
23. The method of claim 1 , wherein the enhanced alpha-helical polypeptide possesses an apparent affinity to human serum proteins of about 10 micromolar or weaker.
24. The method of claim 1 , wherein K d * is defined by the equation
EC
50
′
=
EC
50
+
P
(
n
1
+
K
d
*
EC
50
)
wherein n is 1, EC 50 is an in vitro efficacy measured in a whole cell assay in the absence of any human serum, and EC′ 50 is an in vitro efficacy measured in a whole cell assay in N % human serum wherein P equals (N/100)×(700) micromolar.
25. A polypeptide comprising a cross-linker connecting a first amino acid and a second amino acid of the polypeptide, wherein the polypeptide penetrates cell membranes by an energy-dependent process and binds to a target, and wherein the polypeptide is selected according to the method of claim 1 .